Procedures have been developed in preliminary studies to allow detailed examination of the assembly and structure of gonococcal pili: conditions have been determined to reproducibly obtain reassembled pili, pili sheets and aggregates, disk structures, and three-dimensional crystals. Structure prediction and model building from the amino acid sequence have allowed the identification of an overall structural similarity with tobacco mosaic virus including significant conservation of the area that serves as a nucleotide binding site in the virus. Using electron microscopy, anti-peptide antibodies of predetermined specificity, x-ray diffraction, and computer-aided modeling, we propose to determine the molecular basis for the assembly of pilin subunits, their interaction with DNA, and the recognition of the epithelial cell surface by pili. The areas of the amino acid sequence exposed in the assembled pili will be determined using gold labeled anti-peptide antibodies. Inter-subunit contacts will also be tested by using anti-peptide antibodies and Fab fragments to block different stages of assembly. In combination with antibody labeling, image reconstruction of pilin sheets and disks will allow determination of their subunit composition and packing. Concurrently with electron microscopy studies, re-seeding and other techniques will be used to obtain larger three-dimensional crystals suitable for x-ray structure determination. Crystallographic studies will be pursued, when more suitable crystals become available, to determine the detailed structure of the pilin subunit; this will then be incorporated into the model for inter-subunit packing in the pilus determined by electron microscopy. The structural constraints resulting in the sequence-conserved areas required by the assembly process will be distinguished from those involved in maintaining the postulated DNA binding site and the site required for binding to host epithelial cells. The results of this work should aid the progress of other workers in the rational design of a vaccine against gonorrhea and increase current understanding of the mechanisms of antigenic variation of this major threat to public health. Furthermore, the proposed structural data should be directly applicable to other bacterial species such as Pseudomonas aeruginosa, Moraxella nonliquefaciens, Bacteroides nodosus, and Neisseria meningitidis, all of which produce pili apparently similar in structure and possibly in function to those of the gonococcus.